The objective of the research plan is to study the endocytosis and recycling of cell membrane components at the biochemical and molecular levels, in order to elucidate the molecular mechanisms by which these processes occur. The approach used will be an integrated one, combining techniques of subcellular fractionation, electron microscopy, and protein purification with the in vitro functional reconstitution of steps in the endocytic pathway. Studies will be performed primarily in the Chinese hamster ovary tissue culture line, using the iron transport protein transferrin as a probe of receptor-mediated endocytosis and receptor recycling. Specific goals of the research program include: 1. Isolation, purification and characterization of endocytic membrane elements. 2. Separation of elements involved in internalization from those involved in exocytosis and recycling. 3. Characterization of differences between these membrane subpopulations. 4. Development of in vitro assays that functionally reconstitute steps in the endocytic and recycling pathways. 5. Isolation and characterization of critical components that mediate this functional reconstitution.